Hiw-Wnd pathway regulates the expression and transcriptional activity of the C4da-specific transcription factor Kn.

<p>(A) Overexpressing Wnd attenuates the nuclear Kn expression levels. Representative immunofluorescence images of <i>wt</i> and Wnd-overexpressing (OE Wnd) ddaC neurons labeled with antibodies against Kn (top) and Elav (bottom). Scale bar, 5 µm. (B) Quantification of the immunofluorescence intensity of nuclear Kn normalized to that of nuclear Elav in <i>wt</i>, <i>hiw^ΔN</i>, and OE Wnd neurons. (C) Wnd overexpression down-regulates the promoter activity of the ENaC ion channel <i>pickpocket</i> (<i>ppk</i>), a known target of Kn. Representative ddaC neurons labeled with <i>ppk-eGFP</i> in neurons of the following genotypes: (1) <i>wt</i>; (2) <i>hiw^ΔN</i>; (3) OE Wnd; (4) OE Kn; (5) <i>hiw^ΔN</i>+OE Kn; (6) OE Kn+OE Wnd. Scale bar, 5 µm. (D) Quantification of <i>ppk-eGFP</i> fluorescent intensity in neurons of the following genotypes: (1) <i>wt</i>; (2) <i>hiw^ΔN</i>; (3) OE Wnd; (4) <i>kn^KN1/KN4</i>; (5) OE Kn; (6) <i>hiw^ΔN</i>+OE Kn; (7) OE mCD8RFP+OE Kn; (8) OE Kn+OE Wnd.</p

Transcription factor Fos specifically mediates axonal overgrowth induced by Wnd.

<p>(A) Loss of the <i>Drosophila fos</i>, <i>kay</i>, blocks axonal overgrowth caused by Wnd overexpression. Shown are representative axon terminals of ddaC MARCM clones of following genotypes: (1) <i>wt</i>; (2) overexpressing Wnd with MARCM (OE Wnd); (3) <i>kay¹</i>; (4) overexpressing Wnd in <i>kay¹</i> genetic background with MARCM (OE Wnd+<i>kay¹</i>). Scale bar, 10 µm. (B–B′) <i>kay¹</i> impairs dendritic growth in <i>wt</i> genetic background and exacerbates the dendritic reduction caused by Wnd overexpression. Shown are representative dendrites (B) and tracings (B′) of ddaC MARCM clones of indicated genotypes. Scale bar, 50 µm. (C) Bar charts showing the quantification of axon terminal length (left), total dendrite length (middle), and number of dendrite termini (right).</p

Bimodal Control of Dendritic and Axonal Growth by the Dual Leucine Zipper Kinase Pathway

<div><p>Knowledge of the molecular and genetic mechanisms underlying the separation of dendritic and axonal compartments is not only crucial for understanding the assembly of neural circuits, but also for developing strategies to correct defective dendrites or axons in diseases with subcellular precision. Previous studies have uncovered regulators dedicated to either dendritic or axonal growth. Here we investigate a novel regulatory mechanism that differentially directs dendritic and axonal growth within the same neuron in vivo. We find that the dual leucine zipper kinase (DLK) signaling pathway in <i>Drosophila</i>, which consists of Highwire and Wallenda and controls axonal growth, regeneration, and degeneration, is also involved in dendritic growth in vivo. Highwire, an evolutionarily conserved E3 ubiquitin ligase, restrains axonal growth but acts as a positive regulator for dendritic growth in class IV dendritic arborization neurons in the larva. While both the axonal and dendritic functions of <i>highwire</i> require the DLK kinase Wallenda, these two functions diverge through two downstream transcription factors, Fos and Knot, which mediate the axonal and dendritic regulation, respectively. This study not only reveals a previously unknown function of the conserved DLK pathway in controlling dendrite development, but also provides a novel paradigm for understanding how neuronal compartmentalization and the diversity of neuronal morphology are achieved.</p></div

Wnd mediates the functions of Hiw on dendritic growth.

<p>(A) Loss of <i>wnd</i> blocks dendrite reduction in <i>hiw</i> mutants, and ectopic Wnd restrains dendritic growth. Shown are representative dendrites of ddaC neurons, labeled by <i>ppk-CD4::tdTomato</i>, of the following genotypes: (1) <i>wt</i>; (2) <i>hiw^ΔN</i> homozygotes (<i>hiw</i>); (3) <i>wnd¹/wnd³</i>(<i>wnd</i>); (4) <i>hiw^ΔN</i>; <i>wnd¹/wnd³</i> double mutants (<i>hiw</i>; <i>wnd</i>); (5) overexpressing Wnd by <i>ppkGal4</i> (OE Wnd); (6) overexpressing a kinase dead form (K188A) of Wnd by <i>ppkGal4</i> (OE Wnd^KD). Scale bar, 50 µm. (B) Bar charts showing the quantification of total dendrite length (left) and number of dendrite termini (right). Samples of <i>wt</i> and <i>hiw^ΔN</i> that are used for statistical analysis are the same as those in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001572#pbio-1001572-g001" target="_blank">Figure 1</a>.</p

Regulatory mechanisms underlying dendritic and axonal growth.

<p>(A) Three distinct mechanisms regulating dendritic and axonal growth. Shared mechanisms control dendrite and axon co-growth. Dedicated mechanisms direct compartment-specific growth. Bimodal mechanisms differentially regulate dendritic and axonal growth. (B) A model that postulates the differential control of dendritic and axonal growth by the DLK pathway, which is based on the present study. In this model, DLK plays a dual role in neuron morphogenesis. Up-regulated DLK, caused either by <i>PHR</i> mutations or DLK overactivation, promotes the growth of axon terminals but restricts that of high-order dendritic branches. Such a dichotomous function is the result of signaling divergence into two transcriptional programs that are each dedicated to either dendritic or axonal growth. Fos serves a permissive role in the axonal regulation by DLK, whereas Kn specifically mediates the dendritic regulation by DLK.</p

Hiw differentially regulates dendrite and axon growth in C4da neurons.

<p>(A) Dendrites of the C4da neuron ddaC in <i>hiw^ΔN</i> homozygous mutant larvae are reduced, as compared to <i>wild-type</i> (<i>wt</i>). C4da neurons were labeled by the C4da marker <i>ppk-CD4::tdTomato</i>. Scale bar, 100 µm. (B) Bar charts showing the quantification of total dendrite length (top), number of dendrite termini (bottom) of ddaC in <i>wt</i>, <i>hiw^ΔN</i>, and <i>hiw^ND8</i> larvae. Sample numbers are shown in the bars of the bar charts throughout this article. (C–D) <i>hiw</i> mutant MARCM clones exhibit impaired dendritic growth and overgrowth of axon terminals. (C) Representative dendrites of <i>wt</i> and <i>hiw^ΔN</i> mutant ddaC neurons. Scale bar, 50 µm. (D) Representative axon terminals of a single <i>wt</i> ddaC and a single <i>hiw^ΔN</i> mutant ddaC. The axon terminals of wild-type ddaC clones (green) extend within one segment length of the C4da neuropil (magenta) labeled by <i>ppk-CD4::tdTomato</i>. The axon terminals of <i>hiw^ΔN</i> mutant clones (green) expand over multiple segment lengths of the C4da neuropil (magenta). Scale bar, 10 µm. (E) Quantification of total dendrite length (left) and number of dendrite termini (right) of <i>wt</i> and <i>hiw^ΔN</i> MARCM clones. (F) Quantification of axon terminal length of <i>wt</i> and <i>hiw^ΔN</i> MARCM clones.</p

Kn specifically mediates Hiw regulation of dendritic growth.

<p>(A) <i>hiw</i> and <i>kn</i> interact genetically. Shown are representative dendrites of the following genotypes: (1) <i>hiw^ΔN</i> heterozygote (<i>hiw^ΔN/+</i>); (2) <i>kn ^KN4</i> heterozygote (<i>kn^KN4/+</i>); (3) <i>hiw^ΔN</i> and <i>kn^KN4</i> trans-heterozygote (<i>hiw^ΔN/+</i>; <i>kn^KN4/+</i>). Scale bar, 50 µm. (B) Quantification of total dendrite length of denoted genotypes. <i>wt</i> samples used for statistical analysis are the same as those in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001572#pbio-1001572-g001" target="_blank">Figure 1</a>. (C and C′) Overexpressing Kn partially rescues dendritic defects in <i>hiw^ΔN</i> mutants. Representative dendrites (C) and tracings (C′) of ddaC MARCM clones of following genotypes: (1) <i>wt</i>; (2) <i>hiw^ΔN</i>; (3) overexpressing Kn with MARCM (OE Kn); (4) overexpressing Knot in <i>hiw^ΔN</i> genetic background with MARCM (<i>hiw^ΔN</i>+OE Kn). Scale bar, 50 µm. (D) Quantification of total dendrite length (left) and number of dendrite termini (right). (E) Overexpressing Kn does not alter axon terminal morphology in <i>hiw^ΔN</i> mutants. Shown are representative axon terminals of ddaC MARCM clones of the indicated genotypes. Scale bar, 10 µm. (F) Quantification of the length of axon terminals.</p

Wnd kinase inhibits dendrite growth in C1da neurons expressing ectopic Kn.

<p>(A) Wnd overexpression does not alter dendrite morphology in wild-type C1da neurons, but restrains the dendritic overgrowth caused by ectopic Kn in these neurons. Shown are representative dendrites of C1da neurons ddaD (left) and ddaE (right), labeled by <i>Gal4^2-21</i>/<i>UAS-mCD8::GFP</i>, of the following genotypes: (1) <i>wt</i>; (2) overexpressing Kn by <i>Gal4^2-21</i>(OE Kn); (3) <i>hiw^ΔN</i> homozygotes (<i>hiw</i>); (4) overexpressing Wnd by <i>Gal4^2-21</i> (OE Wnd); (5) overexpressing Kn and Wnd by <i>Gal4^2-21</i> (OE Kn+Wnd); (6) overexpressing Kn and a kinase-dead form of Wnd by <i>Gal4^2-21</i>(OE Kn+Wnd^KD). Scale bar, 50 ⋯µm. Magnified views of the boxed areas are shown on the right for each genotype. (B) Quantification of total dendrite length (left) and number of dendrite termini (right) of ddaEs of denoted genotypes. (C) Wnd kinase specifically down-regulates the expression of <i>UAS-Kn</i>, but not <i>UAS-RedStinger</i> (a nuclear red fluorescent protein) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001572#pbio.1001572-Barolo1" target="_blank">[66]</a> in a posttranscriptional manner. Representative images of ddaEs labeled with antibodies against Kn (top) and RedStinger (bottom) in “OE Kn+Wnd” and “OE Kn+Wnd^KD” using <i>Gal4^2-21</i>. Scale bar, 5 µm. (D) Quantification of immunofluorescence intensity of nuclear Kn normalized to that of RedStinger. Two different <i>Gal4</i> lines, <i>Gal4^2-21</i> (left) and <i>Gal4^21-7</i> (right), were tested in this experiment.</p

The Highwire Ubiquitin Ligase Promotes Axonal Degeneration by Tuning Levels of Nmnat Protein

<div><p>Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Here, using a <em>Drosophila</em> injury model, we have identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw), as an important regulator of axonal and synaptic degeneration. Mutations in <em>hiw</em> strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw: the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat), which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK) MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in <em>hiw</em> mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury.</p> </div

Effects of JNK (bsk) overexpression on NP and axon regeneration, and a summary model.

<p>(A) A 48h NP assay was performed in bsk-overexpressing neurons. The green line indicates a stabilized dendrite. Statistical significance was determined by a Fisher’s exact test and the numbers above the bars indicate numbers of neurons analyzed. *** p<0.001. Control data is from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006503#pgen.1006503.g007" target="_blank">Fig 7D</a>. (B) Axon regeneration assays were performed in neurons overexpressing bsk paired with either mCD8-RFP or Nmnat RNAi. UAS-mCD8-RFP was expressed as a control for Nmnat RNAi to keep the number of UAS-controlled transgenes constant and rule out Gal4 dilution effects. Red arrows mark sites of axon injury. The green star indicates the tip of the converted dendrite. Statistical significance was determined by a Mann-Whitney test. Error bars represent SD. *** p<0.001. (C) A summary model of the results is shown. Axon injury activates the DLK/bsk/fos response pathway. The AP-1 transcription factor fos turns on early injury responses that include Nmnat-mediated NP (indicated by darker cell outline in middle image), microtubule dynamics (short green lines in middle image) and mitochondrial fission. NP is mediated by Nmnat, which, if unchecked dampens subsequent regeneration. Caspases and mitochondrial fission counteract NP. (D) Reduction of caspases, increased bsk or fos, or increased Nmnat result in excess or longer than normal NP. Unbalanced NP dampens regeneration.</p